CN104130315A - Polypeptide for specifically targeting human epidermal growth factor receptor 2 (HER2) protein - Google Patents
Polypeptide for specifically targeting human epidermal growth factor receptor 2 (HER2) protein Download PDFInfo
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- CN104130315A CN104130315A CN201410359780.9A CN201410359780A CN104130315A CN 104130315 A CN104130315 A CN 104130315A CN 201410359780 A CN201410359780 A CN 201410359780A CN 104130315 A CN104130315 A CN 104130315A
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- acid residue
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- cancer
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
Abstract
The invention relates to a polypeptide for specifically targeting a human epidermal growth factor receptor 2 (HER2) protein. The polypeptide is shown in the following general formula 1 of X1X2X3X4X5X6X7RX8YWX9X10X11X12X13X14X15X16X17RX18X19X20X21YX22. The invention also relates to a nucleotide sequence for coding the polypeptide, an expression vector for expressing the polypeptide and a host cell. The invention also relates to the polypeptide and its bivalent or multivalent, a pharmaceutical composition prepared from the polypeptide or its bivalent or multivalent as a targeting polypeptide, a preparation for killing cancer cells and a developing agent, and a use of the polypeptide and its bivalent or multivalent. The polypeptide has HER2 positive cell targeting effects and strong selectivity. The polypeptide can be prepared by a chemical synthesis method and has the advantages of high purity, small molecular weight, strong singularity, no immunogenicity, safety and reliability.
Description
Technical field
The present invention relates to pharmaceutical chemistry field, be specifically related to a peptide species, relate in particular to a kind of polypeptide of special target HER2 albumen and by this peptide derived and can be with the protein bound product of HER2 and aforementioned polypeptides or its derivative product in the purposes of preparing in target HER2 antitumor drug or video picture preparation.
Background technology
Cancer is the major causes of death in the whole world, data presentation: the newly-increased approximately 1,410 ten thousand routine cases of cancers in the whole world in 2012, and cancer mortality number reaches 8,200,000, and by comparison, the data of 2008 are respectively 1,270 ten thousand and 7,600,000.The common cancer of diagnosis is followed successively by lung cancer (1,800,000 in world wide, 13%), mammary cancer (1,700,000,11.9%) and colorectal cancer (1,400,000,9.7%), main lethal cancer is lung cancer (1,600,000,19.4%), liver cancer (800,000,9.1%) and cancer of the stomach (700,000,8.8%).
International cancer research institution estimates according to available data, and due to Regularity Governing World Population Growth and aging, before 2025, the annual newly-increased cancer number of cases in the whole world will be up to 1,930 ten thousand examples.2012, the newly-increased case of cancer over half and the cancer mortality number of whole world sum occurred in low developed area, be respectively 56.8% and 64.9%, and these ratios will further increase in 2025.
At present the methods for the treatment of of malignant tumour mainly contains three kinds of operative treatments, chemotherapy, radiotherapy, and wherein chemotherapy is progressive the fastest methods for the treatment of in oncotherapy in recent years.But chemotherapeutics, in killing tumor cell, has has also killed and wounded human normal cell, and toxic side effect is large.Therefore, anti-tumor drugs targeting and molecular probe research are imperative.
The successful prerequisite of targeting cancer therapy is the target spot of finding treatment accurately.HER2 (human epidermal growth factor receptor 2 has another name called ErbB2) albumen, is the transmembrane glycoprotein receptor tyrosine kinase of 185 kDa, is positioned cytolemma, has the outer ligand binding domain of born of the same parents, single span film district and Bao Nei tyrosine kinase activity district.HER2 participates in signal transduction pathway, causes phosphorylating kinase cascade reaction after activation, causes Growth of Cells and differentiation.Mammary cancer, ovarian cancer, have the overexpression of HER2 in the kinds of tumors such as cancer of the stomach and uterus.HER2 has become the important target for the treatment of cancer.
Polypeptide is easy to a large amount of synthetic, and molecular weight is little, and tissue permeability is strong, can prevent the non-specific uptake of reticuloendothelial system simultaneously.In addition, polypeptide can be chemically modified to change its avidity, electric charge, hydrophobicity, stability and solvability, can use in vivo by optimization amendment repeatedly.In cancer target therapy, polypeptide drug and diagnostic probe demonstrate very strong superiority, are the attractive surrogates of one of antibody.In application aspect, polypeptide coordinates with radionuclide, can be used as probe application in molecular imaging; Some naturally occurring polypeptide have been used as agent delivery; The peptide of reproductive hormone and their derivative can be used for neoplasm targeted therapy.Therefore high-level efficiency finds that valuable active polypeptide has become cancer diagnosis and treated the vital task facing.
Summary of the invention
The object of the present invention is to provide a peptide species, particularly a kind of polypeptide of special target HER2 albumen and by this peptide derived and can be with the protein bound product of HER2 and aforementioned polypeptides or its derived product in the purposes of preparing in target HER2 antitumor drug or video picture preparation.
For reaching this goal of the invention, the present invention by the following technical solutions:
First aspect, the invention provides a kind of peptide of target HER2 albumen, and its aminoacid sequence general formula 1 is:
X
1X
2X
3X
4X
5X
6X
7RX
8YWX
9X
10X
11X
12X
13X
14X
15X
16X
17RX
18X
19X
20X
21YX
22；
In above-mentioned peptide chain, the amino-acid residue of one-letter symbol representative is defined as follows: R is arginine, and Y is Methionin, and W is tryptophane, X
1-22for variable amino-acid residue.
As optimal technical scheme, X
1be amides amino acid or acidic amino acid residue, be preferably l-asparagine or aspartic acid; X
2be alkaline amino acid residue, be preferably Methionin or arginine; X
3be aromatics amino-acid residue, be preferably phenylalanine, tryptophane or tyrosine; X
4be polar amino acid residues, be preferably l-asparagine or arginine; X
5be alkaline amino acid residue, be preferably Methionin or arginine; X
6be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
7be sulfur-containing amino acid or aliphatic amino acid residue, be preferably methionine(Met) or leucine; X
8be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
9be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
10be aliphatic amino acid residue, be preferably L-Ala or leucine; X
11be aliphatic amino acid residue, be preferably leucine or Isoleucine; X
12be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
13be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
14be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
15be amides amino acid or acidic amino acid residue, be preferably l-asparagine or aspartic acid; X
16be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
17be alkaline amino acid residue, be preferably Methionin or arginine; X
18be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
19be aliphatic amino acid residue, be preferably Isoleucine or leucine; X
20be sulfur-containing amino acid or alkaline amino acid residue, be preferably methionine(Met) or arginine; X
21be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
22be aliphatic amino acid or polar amino acid residues, be preferably leucine, arginine, l-asparagine or aspartic acid.
As optimal technical scheme, the aminoacid sequence of peptide of the present invention is shown in SEQ ID NO.1 or SEQ ID NO.2.
Second aspect, the present invention also provides a kind of DNA fragmentation, the aminoacid sequence of its peptide that comprises coding the invention described above general formula 1.
As optimal technical scheme, the aminoacid sequence of the peptide that described DNA fragmentation comprises coding the invention described above SEQ ID NO.1 or SEQ ID NO.2.
The third aspect, the present invention also provides a kind of expression vector, and the encoding amino acid sequence that comprises at least one copy is the DNA fragmentation described in the second aspect present invention of peptide shown in general formula 1.
As optimal technical scheme, expression vector of the present invention, the encoding amino acid sequence that comprises at least one copy is the DNA fragmentation described in the second aspect present invention of peptide shown in SEQ ID NO.1 or SEQ ID NO.2.
Fourth aspect, the present invention also provides a kind of protokaryon or eukaryotic host cell, and this host cell contains just like the expression vector described in third aspect present invention.
The 5th aspect, the present invention also provides a kind of bivalent or multivalent, is assembled by peptide described in peptide described in the general formula 1 of first aspect present invention and SEQ ID NO.1 or SEQ ID NO.2.
Bivalent as above or multivalent are the characteristics with the tumour cell of the target HER2 positive.
As optimal technical scheme, bivalent of the present invention or multivalent are by connecting the covalently bound formation of molecule or by mixing with polymer, non-covalent being connected to form.
Preferably, described polymer is polyoxyethylene glycol (PEG) or cyclodextrin.
The 6th aspect, the present invention also further provides a kind of pharmaceutical composition, the aminoacid sequence that comprises first aspect present invention be the bivalent of peptide described in peptide described in general formula 1, SEQ ID NO.1, SEQ ID NO.2 or described peptide or multivalent as target polypeptide, and can kill and wound the preparation of cancer cells.
As optimal technical scheme, peptide of the present invention, bivalent or multivalent, as target polypeptide, are puted together mutually or mix with the preparation that can kill and wound cancer cells.
Preferably, described preparation is any one in chemicals, bio-pharmaceutical, Nano medication, radiopharmaceuticals, photo-thermal therapy or the optical dynamic therapy medicine that can kill and wound cancer cells or the carrier that wraps up these medicines;
Further preferably, described preparation is any one in alkylating agent, antimetabolite, antitumor natural drug, antitumor antibiotics, hormone and metal complex or tumour radiotherapy target marker.
Further preferably, described carrier is nano material, any one in liposome or oiliness compound, or the mixture being made up of multiple oiliness compound.
The present invention adopts the macromolecular material such as bivalent or multivalent and nano material, liposome of peptide described in general formula 1, SEQ ID NO.1 or SEQ ID NO.2 and described peptide is puted together, and peptide, bivalent or the multivalent the present invention relates to can make the compound of puting together rear generation in body, more stably be transported to target cell.Peptide, bivalent or the multivalent the present invention relates to also can mix mutually with the mixture of oiliness compound or multiple oiliness compound, the peptide the present invention relates to also can make gained to mixture in body, be more stably transported to target cell.
The 7th aspect, the present invention also further provides another pharmaceutical composition, and described pharmaceutical composition comprises that the aminoacid sequence of first aspect present invention is peptide described in peptide described in general formula 1, SEQ ID NO.1, SEQ ID NO.2 or bivalent or the multivalent of described peptide; With video picture preparation.
Preferably, described peptide, bivalent or multivalent are puted together mutually or mix with video picture preparation.
Preferably, described video picture preparation is any one in radionuclide, radioisotope labeling thing or molecular image preparation.
Eight aspect, it is peptide described in peptide described in general formula 1, SEQ ID NO.1, SEQ ID NO.2 or bivalent or the purposes of multivalent in the medicine for the preparation for the treatment of, prevention or diagnosing cancer or video picture preparation of described peptide that the present invention also provides the aminoacid sequence of first aspect present invention.
As optimal technical scheme, cancer of the present invention is the cancer that HER2 crosses expression.
Preferably, described cancer is mammary cancer, lung cancer, cancer of the stomach, liver cancer, colon and rectum carcinoma, the esophageal carcinoma, leukemia, bladder cancer, cervical cancer
Peptide of the present invention has the effect of target HER2 albumen, can be used as target head increases medicine or the carrier that is loaded with medicine as the content in HER2 positive cell such as nano material, liposome, then adds pharmaceutically acceptable auxiliary material or adjuvant is made novel more effective targeted anticancer medicine.
Compared with prior art, the present invention has following beneficial effect:
The Toplink the present invention relates to plays targeting to HER2 positive cell, and selectivity is strong, and the peptide the present invention relates to can adopt the method for chemosynthesis to prepare, and purity is high, and molecular weight is little, high specificity, and non-immunogenicity, safe and reliable.
Brief description of the drawings
Fig. 1 is SEQ ID NO.1, SEQ ID NO.2 and the antibody keying action to people HER2 positive breast cancer cell (SKBR3 clone) respectively that fluidic cell method detects fluorescein isothiocyanate (FITC) mark;
Wherein A is control group, and B is Anti-HER 2, and C is SEQ ID NO.1, and D is SEQ ID NO.2, and E is combination rate histogram.
Fig. 2 is SEQ ID NO.1, SEQ ID NO.2 and the antibody keying action to people HER2 negative breast cancer cell (MCF-7 clone) respectively that fluidic cell method detects FITC mark;
Wherein A is control group, and B is Anti-HER 2, and C is SEQ ID NO.1, and D is SEQ ID NO.2.
Fig. 3 is SEQ ID NO.1, SEQ ID NO.2 and the antibody keying action to the negative embryonic kidney normal cell of people HER2 (293A clone) respectively that fluidic cell method detects FITC mark;
Wherein A is control group, and B is Anti-HER 2, and C is SEQ ID NO.1, and D is SEQ ID NO.2.
Fig. 4 is SEQ ID NO.1, the SEQ ID NO.2 keying action to people HER2 positive breast cancer cell (SKBR3 clone) respectively that immunofluorescence method detects FITC mark;
Wherein, nucleus hochest reagent dyeing; A-D is the cell that SEQ ID NO.1 polypeptide and hochest processed, and wherein A is the binding site of SEQ ID NO.1 polypeptide, is mainly present in cytolemma, and B is nucleus, and C is the whole cell of bright field, the superimposed figure that D is A-C; E-H is the cell that SEQ ID NO.2 polypeptide and hochest processed, and wherein E is the binding site of SEQ ID NO.2 polypeptide, is mainly present in cytolemma, and F is nucleus, and G is the whole cell of bright field, the superimposed figure that H is E-G.
Fig. 5 is SEQ ID NO.1, the SEQ ID NO.2 keying action to people HER2 negative breast cancer cell (MCF-7 clone) respectively that immunofluorescence method detects FITC mark;
Wherein, nucleus hochest reagent dyeing; A-D is the cell that SEQ ID NO.1 polypeptide and hochest processed, and wherein A is the binding site of SEQ ID NO.1 polypeptide, and B is nucleus, and C is the whole cell of bright field, the superimposed figure that D is A-C; E-H is the cell that SEQ ID NO.2 polypeptide and hochest processed, and wherein E is the binding site of SEQ ID NO.2 polypeptide, and F is nucleus, and G is the whole cell of bright field, the superimposed figure that H is E-G.
Fig. 6 is SEQ ID NO.1, the SEQ ID NO.2 keying action to the negative embryonic kidney normal cell of people HER2 (293A clone) respectively that immunofluorescence method detects FITC mark;
Wherein, nucleus hochest reagent dyeing; A-D is the cell that SEQ ID NO.1 polypeptide and hochest processed, and wherein A is the binding site of SEQ ID NO.1 polypeptide, and B is nucleus, and C is the whole cell of bright field, the superimposed figure that D is A-C; E-H is the cell that SEQ ID NO.2 polypeptide and hochest processed, and wherein E is the binding site of SEQ ID NO.2 polypeptide, and F is nucleus, and G is the whole cell of bright field, the superimposed figure that H is E-G.
Fig. 7 is that surface plasma resonance (SPR) method detects SEQ ID NO.1 (A), SEQ ID NO.2 (B) keying action of the people HER2 albumen to different concns respectively.
Aminoacid sequence:
SEQ?ID?NO.1：NKFNKGMRGYWGALGGGNGKRGIRGYD
SEQ?ID?NO.2：NKFNKGMRGYWGALGGGNGKRGIMGYD
Embodiment
Further describe the present invention below in conjunction with specific embodiment, it will be understood by those skilled in the art that described embodiment is only intended as illustrative explanation the present invention, instead of intention limits the scope of the invention.Scope of the present invention is specifically limited by accompanying claim.
Synthesizing of embodiment 1 polypeptide of the present invention
1) laboratory apparatus and material
Dimethyl formamide (DMF), piperidines, resin, methylene dichloride (DCM), ninhydrin reaction reagent (triketohydrindene hydrate, Vc, phenol), tetramethyl-urea hexafluorophosphate (HBTU), hexahydropyridine (piperidines), tri isopropyl silane TIS, dithioglycol (EDT), anhydrous diethyl ether, trifluoroacetic acid (TFA), N-methylmorpholine (NMM), methyl alcohol, each seed amino acid, Solid-phase Polypeptide composite tube.
2) solution preparation
Deprotection solvent---hexahydropyridine: DMF=1:4
Reaction solution---NMM:DMF=1:24
Lysate---TFA (92.5%), TIS (2.5%), EDT (2.5%), H
2o (2.5%)
Triketohydrindene hydrate test fluid---triketohydrindene hydrate: Vc: phenol=1:1:1
3) experimental procedure
Weigh resin and also put in Solid-phase Polypeptide composite tube (hereinafter to be referred as reactor), more than adding appropriate DMF swelling half an hour.Take out DMF, carry out Fmoc protective reaction with deprotection liquid, be placed in shaking table upper 10 minute.Take out and protect liquid, with DMF, DCM washing 3 times, the resin that takes a morsel from reactor (approximately 5～10mg), in test tube, is used washing with alcohol 2 times, and ninhydrin method detects and record color, prepares to feed intake, and enters amino acid condensation reaction.Get corresponding amino acid, HBTU (amino acid: HBTU=1:1) according to the aminoacid sequence order of SEQ ID NO.1, SEQ ID NO.2 peptide respectively, with reaction solution dissolving, put in reactor stirring reaction.After 1-2 hour, take a morsel resin from reactor in test tube, use washing with alcohol 2 times, ninhydrin method detects.Take out the liquid in reactor, respectively wash 2 times with DMF, DCM, obtain the peptide resin after first amino acid condensation.Gained peptide resin is repeated to above " Fmoc removes protection---amino acid condensation " reactions steps, and complete to last amino acid reaction, obtaining sequence number is the peptide of SEQ ID NO.1 and SEQ ID NO.2.After completion of the reaction, the each washing resin of DMF, DCM 2-3 time, methyl alcohol washes twice, continues to drain 15-20 minute.In reactor, take out the partial peptide resin that synthesize, at room temperature cracking two hours in lysate (lysate elder generation ice bath 20 minutes).By after resin filter, steam instrument evaporate to dryness in revolving, wash 3 times with anhydrous diethyl ether (ice bath).Thick peptide uses preparative reversed-phase HPLC purifying, uses HPLC to detect purity >90%.The pure peptide obtaining uses mass spectrum (MS, eLectrospray) qualification.
After synthetic to last peptide, take out part peptide resin and add fluorescein isothiocyanate (FITC) fluorescent mark.First Fmoc-e-Acp-OH is linked on polypeptide by amino acid coupling method, then gets appropriate HBTU and FITC is dissolved in fluorescence coupling solvent, after 3-5 hours, triketohydrindene hydrate is surveyed solution testing.After mark success, adopt above-mentioned same method to carry out cracking, purifying and qualification.
The relevant physicochemical character of SEQ ID NO.1, SEQ ID NO.2 peptide is as shown in table 1.
Table 1
Experimental example 1 fluidic cell method detects SEQ ID NO.1, the SEQ ID NO.2 keying action to people HER2 positive breast cancer cell respectively
One, experimental technique
Collector's mammary cancer HER2 overexpression cell line SKBR3, is suspended in containing in the RPMI1640 nutrient solution of 10% heat-inactivated fetal bovine serum, and cell density is at 1x10
6/ mL left and right, is sub-packed in the EP pipe of four 1.5mL, 200 μ L/ pipes.Add respectively SEQ ID NO.1, SEQ ID NO.2, the Anti-HER 2 (ebioscience of fluorescein isothiocyanate (FITC) mark, BMS120FI), ultimate density is 50 μ moL/L, and control group is used with the 0.01mM PBS (phosphoric acid buffer pH7.4) of polypeptide equivalent and replaced polypeptide.Lucifuge ice bath was hatched after 30 minutes, and centrifugal 4 minutes collecting cells of 1000g add 1mL PBS to clean, and after repeated washing 2 times, add 500 μ L PBS, mix, and use fluidic cell method fluorescence intensity and combining ratio.
Two, experimental result
As seen from Figure 1, SEQ ID NO.1, SEQ ID NO.2 peptide can be in conjunction with human breast carcinoma HER2 high expressing cells, illustrate that peptide of the present invention uses HER2 positive tumor cell is had to affinity interaction separately, and the polypeptide that can be used as target HER2 uses.
Experimental example 2 fluidic cell methods detect SEQ ID NO.1, the SEQ ID NO.2 keying action to people HER2 negative breast cancer cell respectively
One, experimental technique
Collector's mammary cancer HER2 down-regulated express cell strain MCF-7, is suspended in containing in the H-DMEM nutrient solution of 10% heat-inactivated fetal bovine serum, and cell density is at 1x10
6/ mL left and right, is sub-packed in four 1.5 mL EP pipes.200 μ L/ pipes.Add respectively SEQ ID NO.1, SEQ ID NO.2, the Anti-HER 2 (ebioscience of fluorescein isothiocyanate (FITC) mark, BMS120FI), ultimate density is 50 μ mol/L, and control group is used with the 0.01 mM PBS (phosphoric acid buffer pH7.4) of polypeptide equivalent and replaced polypeptide.Lucifuge ice bath was hatched after 30 minutes, and centrifugal 4 minutes collecting cells of 1000 g add 1 mL PBS to clean, and after repeated washing 2 times, add 500 μ L PBS, mix, and use fluidic cell method fluorescence intensity and combining ratio.
Two, experimental result
As seen from Figure 2, a little less than the combination of SEQ ID NO.1, SEQ ID NO.2 peptide and the low expression human breast cancer cell of HER2 MCF-7, illustrate that peptide of the present invention is to the selective keying action of HER2, the polypeptide that can be used as special target HER2 uses.
Experimental example 3 fluidic cell methods detect SEQ ID NO.1, SEQ ID NO.2 respectively to the Normocellular keying action of the negative embryonic kidney of people HER2
One, experimental technique
Collector's embryonic kidney HER2 down-regulated express cell strain 293A, is suspended in containing in the H-DMEM nutrient solution of 10% heat-inactivated fetal bovine serum, and cell density is at 1x10
6/ mL left and right, is sub-packed in four 1.5 mL EP pipes.200 μ L/ pipes.Add respectively SEQ ID NO.1, SEQ ID NO.2, the Anti-HER 2 (ebioscience of fluorescein isothiocyanate (FITC) mark, BMS120FI), ultimate density is 50 μ mol/L, and control group is used with the 0.01 mM PBS (phosphoric acid buffer pH7.4) of polypeptide equivalent and replaced polypeptide.Lucifuge ice bath was hatched after 30 minutes, and centrifugal 4 minutes collecting cells of 1000 g add 1 mL PBS to clean, and after repeated washing 2 times, add 500 μ L PBS, mix, and use fluidic cell method fluorescence intensity and combining ratio.
Two, experimental result
As seen from Figure 3, the keying action of SEQ ID NO.1, SEQ ID NO.2 peptide and the low expression human embryo kidney (HEK) of HER2 normal cell 293A cell is very micro-, illustrates that peptide of the present invention is to the selective keying action of HER2, and the polypeptide that can be used as special target HER2 uses.
Experimental example 4 immunofluorescence methods detect SEQ ID NO.1, the SEQ ID NO.2 keying action to people HER2 positive breast cancer cell respectively
One, experimental technique
Human breast carcinoma HER2 overexpression cell line SKBR3 is suspended in containing in the RPMI1640 nutrient solution of 10% heat-inactivated fetal bovine serum, is inoculated in three confocol capsules with the density of 3000-5000/ware.Cultivate after 24 hours, substratum in exhaustion capsule, then add respectively the SEQ ID NO.1 of the 50 μ mol/L that contain fluorescein isothiocyanate (FITC) mark, the substratum 200 μ L of SEQ ID NO.2 polypeptide, control group with containing with the substratum of the PBS (phosphoric acid buffer pH7.4) of polypeptide equivalent, nucleus hochest reagent dyeing, uses and dilutes according to 1:200.Lucifuge ice bath is hatched 30 minutes.PBS cleans, and repeats to wash after 3 times, adds 200 μ L PBS, uses laser confocal microscope observation fluorescent signal.
Two, experimental result
As seen from Figure 4, SEQ ID NO.1, SEQ ID NO.2 peptide can act on the surface of human breast carcinoma HER2 positive cell, illustrate that peptide of the present invention uses HER2 positive tumor cell is had to keying action separately, and the polypeptide that can be used as target HER2 uses.
Experimental example 5 immunofluorescence methods detect SEQ ID NO.1, the SEQ ID NO.2 keying action to people HER2 negative breast cancer cell respectively
One, experimental technique
Human breast carcinoma HER2 down-regulated express cell strain MCF-7 is suspended in containing in the H-DMEM nutrient solution of 10% heat-inactivated fetal bovine serum, is inoculated in three confocol capsules with the density of 3000-5000/ware.Cultivate after 24 hours, substratum in exhaustion capsule, then add respectively the SEQ ID NO.1 of the 50 μ mol/L that contain fluorescein isothiocyanate (FITC) mark, the substratum 200 μ L of SEQ ID NO.2 polypeptide, control group with containing with the substratum of the PBS (phosphoric acid buffer pH7.4) of polypeptide equivalent, nucleus hochest reagent dyeing, uses and dilutes according to 1:200.Lucifuge ice bath is hatched 30 minutes.PBS cleans, and repeats to wash after 3 times, adds 200 μ L PBS, uses laser confocal microscope observation fluorescent signal.
Two, experimental result
As seen from Figure 5, SEQ ID NO.1, SEQ ID NO.2 peptide and human breast carcinoma HER2 negative cells do not have combination, illustrate that peptide of the present invention is selectivity specific binding to HER2 positive tumor cell, and the polypeptide that can be used as special target HER2 uses.
Experimental example 6 immunofluorescence methods detect SEQ ID NO.1, SEQ ID NO.2 respectively to the Normocellular keying action of the negative embryonic kidney of people HER2
One, experimental technique
The negative human embryo kidney (HEK) normal cell of HER2 strain 293A is suspended in containing in the H-DMEM nutrient solution of 10% heat-inactivated fetal bovine serum, is inoculated in three confocol capsules with the density of 3000-5000/ware.Cultivate after 24 hours, substratum in exhaustion capsule, then add respectively the SEQ ID NO.1 of the 50 μ mol/L that contain fluorescein isothiocyanate (FITC) mark, the substratum 200 μ L of SEQ ID NO.2 polypeptide, control group with containing with the substratum of the PBS (phosphoric acid buffer pH7.4) of polypeptide equivalent, nucleus hochest reagent dyeing, uses and dilutes according to 1:200.Lucifuge ice bath is hatched 30 minutes.PBS cleans, and repeats to wash after 3 times, adds 200 μ L PBS, uses laser confocal microscope observation fluorescent signal.
Two, experimental result
As seen from Figure 6, the negative embryonic kidney normal cell of SEQ ID NO.1, SEQ ID NO.2 peptide and people HER2 does not have combination, illustrates that peptide of the present invention is selectivity specific binding to HER2 positive tumor cell, and the polypeptide that can be used as special target HER2 uses.
Experimental example 7 surface plasma resonances (SPR) method detects SEQ ID NO.1, the SEQ ID NO.2 keying action to people HER2 albumen respectively
One, experimental technique
By the SEQ ID NO.1 of 1 mg/mL, SEQ ID NO.2 peptide and 0.01 mM PBS point to chip, overnight incubation under 4 DEG C of wet condition, then clean ten minutes with 0.1 mM PBS, 0.01 mM PBS cleans ten minutes, finally use washed with de-ionized water ten minutes, and repeat once, immerse containing in 0.01 mMPBS of 5% milk, overnight incubation under 4 DEG C of conditions, then cleans ten minutes with 0.1 mM PBS, and 0.01 mM PBS cleans ten minutes, finally use washed with de-ionized water ten minutes, and repeat once, dry up upper machine with nitrogen.Moving phase is passed through the people HER2 albumen of 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL and 20 μ g/mL, record analysis spr signal successively.
Two, experimental result
As seen from Figure 7, the spr signal of SEQ ID NO.1, SEQ ID NO.2 peptide, along with the increase of people HER2 protein concentration strengthens gradually, illustrates that peptide of the present invention has strong keying action to HER2, and the polypeptide that can be used as target HER2 uses.
The result of experimental example 1-7 shows, on cell levels, fluidic cell is learned and two kinds of experiment tests of immunofluorescence technique all show that SEQ ID NO.1, SEQ ID NO.2 polypeptide are to be specially combined with the breast cancer cell of the HER2 positive, and be incorporated into the surface of cell membrane of HER2 protein expression, and there is no obvious combination with breast cancer cell and the embryonic kidney normal cell of HER2 feminine gender; On molecular level, surface plasma resonance (SPR) experiment has confirmed that SEQ ID NO.1, SEQ ID NO.2 polypeptide all have combination with HER2 albumen too, and along with the raising of HER2 protein concentration, binding signal is stronger.
Can draw from experimental example 1-7, peptide of the present invention has the characteristic of target HER2 positive tumor cell, thereby in actual applications, can be using peptide of the present invention as target polypeptide, put together mutually or mix with the preparation that can kill and wound cancer cells, for the targeted therapy of tumour.
Applicant's statement, the present invention illustrates processing method of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned processing step, does not mean that the present invention must rely on above-mentioned processing step and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, and the selections of the equivalence replacement to the selected raw material of the present invention and the interpolation of ancillary component, concrete mode etc., within all dropping on protection scope of the present invention and open scope.
Claims (10)
1. a peptide for special target HER2 albumen, is characterized in that, its general formula is:
X
1X
2X
3X
4X
5X
6X
7RX
8YWX
9X
10X
11X
12X
13X
14X
15X
16X
17RX
18X
19X
20X
21YX
22
Wherein, R is arginine, and Y is Methionin, and W is tryptophane, X
1-22for variable amino-acid residue;
Preferably, X
1be amides amino acid or acidic amino acid residue, be preferably l-asparagine or aspartic acid; X
2be alkaline amino acid residue, be preferably Methionin or arginine; X
3be aromatics amino-acid residue, be preferably phenylalanine, tryptophane or tyrosine; X
4be polar amino acid residues, be preferably l-asparagine or arginine; X
5be alkaline amino acid residue, be preferably Methionin or arginine; X
6be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
7be sulfur-containing amino acid or aliphatic amino acid residue, be preferably methionine(Met) or leucine; X
8be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
9be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
10be aliphatic amino acid residue, be preferably L-Ala or leucine; X
11be aliphatic amino acid residue, be preferably leucine or Isoleucine; X
12be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
13be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
14be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
15be amides amino acid or acidic amino acid residue, be preferably l-asparagine or aspartic acid; X
16be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
17be alkaline amino acid residue, be preferably Methionin or arginine; X
18be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
19be aliphatic amino acid residue, be preferably Isoleucine or leucine; X
20be sulfur-containing amino acid or alkaline amino acid residue, be preferably methionine(Met) or arginine; X
21be polare Aminosaeren or aliphatic amino acid residue, be preferably glycine or L-Ala; X
22be aliphatic amino acid or polar amino acid residues, be preferably leucine, arginine, l-asparagine or aspartic acid.
2. peptide according to claim 1, is characterized in that, the aminoacid sequence of described peptide is shown in SEQID NO.1 or SEQ ID NO.2.
3. a DNA fragmentation, is characterized in that, the nucleotide sequence that it comprises peptide described in coding claim 1-2 any one.
4. an expression vector, is characterized in that, the DNA fragmentation as claimed in claim 3 that described expression vector contains at least one copy.
5. a host cell, is characterized in that, described host cell contains expression vector claimed in claim 4.
6. the bivalent or the multivalent that form according to the peptide described in claim 1-2 any one, is characterized in that, described bivalent or multivalent have the characteristic of target HER2 positive tumor cell;
Preferably, described bivalent or multivalent are by connecting the covalently bound formation of molecule;
Preferably, described bivalent or multivalent are by mixing with polymer, non-covalent being connected to form;
Preferably, described polymer is polyoxyethylene glycol (PEG) or cyclodextrin.
7. a pharmaceutical composition, is characterized in that,
Described pharmaceutical composition comprises the peptide described in claim 1-2 any one and can kill and wound the preparation of cancer cells;
Preferably, described pharmaceutical composition comprises bivalent claimed in claim 6 or multivalent and the preparation that can kill and wound cancer cells;
Preferably, described peptide, bivalent or multivalent, as target polypeptide, are puted together mutually or mix with the preparation that can kill and wound cancer cells;
Preferably, described preparation is any one in chemicals, bio-pharmaceutical, Nano medication, radiopharmaceuticals, photo-thermal therapy or the optical dynamic therapy medicine that can kill and wound cancer cells or the carrier that wraps up these medicines;
Further preferably, described preparation is any one in alkylating agent, antimetabolite, antitumor natural drug, antitumor antibiotics, hormone, metal complex or tumour radiotherapy target marker;
Further preferably, described carrier is nano material, any one in liposome or oiliness compound, or the mixture being made up of multiple oiliness compound.
8. a pharmaceutical composition, is characterized in that, described pharmaceutical composition comprises peptide and the video picture preparation described in claim 1-2 any one;
Preferably, described pharmaceutical composition comprises bivalent claimed in claim 6 or multivalent and video picture preparation;
Preferably, described peptide, bivalent or multivalent are puted together mutually or mix with video picture preparation;
Preferably, described video picture preparation is any one in radionuclide, radioisotope labeling thing or molecular image preparation.
9. the purposes of peptide, bivalent or the multivalent described in claim 1-2 any one or 6 in the medicine for the preparation for the treatment of, prevention or diagnosing cancer or video picture preparation.
10. purposes according to claim 9, is characterized in that, described cancer is the cancer that HER2 crosses expression;
Preferably, described cancer is any one in mammary cancer, lung cancer, cancer of the stomach, liver cancer, colon and rectum carcinoma, the esophageal carcinoma, leukemia, bladder cancer, cervical cancer or nasopharyngeal carcinoma.
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| CN201410359780.9A CN104130315B (en) | 2014-07-25 | 2014-07-25 | A kind of polypeptide of special target HER2 albumen |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105085631A (en) * | 2015-09-14 | 2015-11-25 | 国家纳米科学中心 | Polypeptide capable of specifically targeting HER2 protein and application of polypeptide |
| CN105085632A (en) * | 2015-09-14 | 2015-11-25 | 国家纳米科学中心 | Specific EGFR (epidermal growth factor receptor)-protein-targeted polypeptide and application thereof |
| CN105198964A (en) * | 2015-10-12 | 2015-12-30 | 国家纳米科学中心 | Tumor targeted polypeptide, and preparation method and application thereof |
| CN105330725A (en) * | 2015-11-06 | 2016-02-17 | 国家纳米科学中心 | Polypeptide with pH response and human tumor vessel marker VEGFR2 targeting functions and application of polypeptide |
| CN105693860A (en) * | 2016-03-03 | 2016-06-22 | 国家纳米科学中心 | Specific HER2 protein targeted polypeptide and application thereof |
| WO2017113444A1 (en) * | 2015-12-31 | 2017-07-06 | 杭州阿诺生物医药科技股份有限公司 | Her-2 polypeptide for tumor, composition, preparation method and application |
| CN108752416A (en) * | 2018-06-29 | 2018-11-06 | 烟台智本知识产权运营管理有限公司 | The preparation method of amino acid polypeptide |
| CN111548419A (en) * | 2020-04-26 | 2020-08-18 | 国家纳米科学中心 | DDR2 targeting polypeptide and application thereof |
| CN112358531A (en) * | 2020-11-09 | 2021-02-12 | 国家纳米科学中心 | Polypeptide targeting HER2 protein and application thereof |
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| CN105085631A (en) * | 2015-09-14 | 2015-11-25 | 国家纳米科学中心 | Polypeptide capable of specifically targeting HER2 protein and application of polypeptide |
| CN105085631B (en) * | 2015-09-14 | 2019-01-25 | 国家纳米科学中心 | A kind of polypeptide of special target HER2 albumen and its application |
| CN105085632B (en) * | 2015-09-14 | 2019-01-25 | 国家纳米科学中心 | A kind of polypeptide of special target EGFR albumen and its application |
| CN105198964B (en) * | 2015-10-12 | 2019-01-25 | 国家纳米科学中心 | A kind of cancer target polypeptide, preparation method and application |
| CN105198964A (en) * | 2015-10-12 | 2015-12-30 | 国家纳米科学中心 | Tumor targeted polypeptide, and preparation method and application thereof |
| CN105330725A (en) * | 2015-11-06 | 2016-02-17 | 国家纳米科学中心 | Polypeptide with pH response and human tumor vessel marker VEGFR2 targeting functions and application of polypeptide |
| WO2017113444A1 (en) * | 2015-12-31 | 2017-07-06 | 杭州阿诺生物医药科技股份有限公司 | Her-2 polypeptide for tumor, composition, preparation method and application |
| CN105693860A (en) * | 2016-03-03 | 2016-06-22 | 国家纳米科学中心 | Specific HER2 protein targeted polypeptide and application thereof |
| CN105693860B (en) * | 2016-03-03 | 2019-08-27 | 国家纳米科学中心 | The polypeptide of selectively targeted HER2 albumen and its application |
| CN108752416A (en) * | 2018-06-29 | 2018-11-06 | 烟台智本知识产权运营管理有限公司 | The preparation method of amino acid polypeptide |
| CN111548419A (en) * | 2020-04-26 | 2020-08-18 | 国家纳米科学中心 | DDR2 targeting polypeptide and application thereof |
| CN111548419B (en) * | 2020-04-26 | 2021-11-16 | 国家纳米科学中心 | DDR2 targeting polypeptide and application thereof |
| CN112358531A (en) * | 2020-11-09 | 2021-02-12 | 国家纳米科学中心 | Polypeptide targeting HER2 protein and application thereof |
| CN112358531B (en) * | 2020-11-09 | 2022-05-27 | 国家纳米科学中心 | Polypeptide targeting HER2 protein and application thereof |
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